Insights into the missing apiosylation step in flavonoid apiosides biosynthesis of Leguminosae plants

Apiose is a natural pentose containing an unusual branched-chain structure. Apiosides are bioactive natural products widely present in the plant kingdom. However, little is known on the key apiosylation reaction in the biosynthetic pathways of apiosides. In this work, we discover an apiosyltransferase GuApiGT from Glycyrrhiza uralensis. GuApiGT could efficiently catalyze 2″-O-apiosylation of flavonoid glycosides, and exhibits strict selectivity towards UDP-apiose. We further solve the crystal structure of GuApiGT, determine a key sugar-binding motif (RLGSDH) through structural analysis and theoretical calculations, and obtain mutants with altered sugar selectivity through protein engineering. Moreover, we discover 121 candidate apiosyltransferase genes from Leguminosae plants, and identify the functions of 4 enzymes. Finally, we introduce GuApiGT and its upstream genes into Nicotiana benthamiana, and complete de novo biosynthesis of a series of flavonoid apiosides. This work reports an efficient phenolic apiosyltransferase, and reveals mechanisms for its sugar donor selectivity.

The manuscript entitled 'Discovery, mechanisms, and engineering of the missing apiosylation step in the biosynthesis of flavonoid aposides in Leguminosae plants' reported an apiosyltransferase GuApiGT from medicinal plant G. uralensis.Biochemical characterization supported that GuApiGT specifically utilizes UDP-apiose as a sugar donor and catalyzes 2''-O-apiosylation of flavonoid glycosides.The binding site for UDP-apiose was determined through structural determination, theoretical calculations, and metagenesis analysis.This enzyme also shows broad substrate promiscuity, it accepts 37 glycosides of flavonoids, liganans, and coumarins.Several new compounds were generated and may show bioactivity.The authors successfully performed de novo biosynthesis to produce various flavonoid apiosides in tobacco.This work discovered 121 candidate apiosyltransferase genes from Leguminosae plants and determined the first crystal structure of apiosyltransferase.The key apiose binding motif (RLGSDH) was identified, providing important information for engineering of glycosyltransferases.The results possess significant novelty, the presentation is readable.Below are some of my major and minor concerns regarding the article: Major Concern: 1.The authors mention many times that 'no reported UGTs could accept UDP-apiose as the sugar donor'; however, there is a research article, Identification of an apiosyltransferase in the plant pathogen Xanthomonas pisi (PLoS ONE 13 (10) 2018), reported the first evidence of an apiosyltransferase (XpApiT).Although the sugar acceptor of XpApiT was unknown, indirect activity assay (UDP-Glo) and in microbe co-expression supported that XpApiT specifically utilizes UDP-apiose as sugar donor.Does XpApiT possess the conserved apiose-binding residues or motifs, such as RLGSDH motif?Please rephrase the relevant sentences, 'no reported……', into more appropriate ones.2. GuApiGT catalyzes the formation of glycosidic bond between O-2'' of the glucose residue (Compound 2) and C-1''' of the Api moiety (UDP-Api).However, in the docking model shown in Fig. 4e, O-2'' of the glucose residue seems to be far away from C-1''' of the Api moiety.Is this a reasonable distance for catalysis?Please label the distance between O-2'' of the glucose residue (Compound 2) and C-1''' of the Api moiety (UDP-Api).3.In Fig. 4g, the authors show QM/MM optimized geometry of transition state (TS), where O-2'' of the glucose residue is covalent bonded with H18.In general, D115 facilitates H18 to deprotonate the hydroxyl group of O-2''.The covalent bond between O-2'' and C-1''' should not be the TS.Furthermore, the authors did not describe the roles of H18 and D115.Are these two residues conserved among the GuApiGT homologues?Please regenerate Fig. g and describe the function of H18 and D115.
Minor Concern: 1. Page 3: Line 51, 'form' should be 'from'.2. Page11: Line208, the authors should tell readers what and where the PSPG box is. 3.In Fig. 4f, the authors only show a sugar donor, UDP-Api.The figure legend does not match to the figure.4. Fig. 4g is not very clear.The structure of Api appears to be incorrect.Please generate a new one to make it more accessible.5. GuApiGT catalyzes the formation of glycosidic bond between O-2'' of the glucose residue (Compound 2) and C-1''' of the Api moiety (UDP-Api).The labeling of C1' and O5 in Fig. f is confusing.Please use C1''' and O2'' instead of C1' and O5 in Fig. f and the text.6.The 'Number of atoms' of Sb3GT1 375S/Q377H in Table 8 does not match to the crystal structure.Please revise Table 8.
Reviewer #3 (Remarks to the Author): Recommendation: Accept after major revisions.
The paper presented by Ye and colleagues delivers a comprehensive investigation of a previously unreported apiosyltransferase, GuApiGT, from Glycyrrhiza uralensis.Their study elegantly elucidates the mechanism of GuApiGT's role in catalyzing the 2''-O-apiosylation of flavonoid glycosides.This has been achieved by analyzing the enzyme's structure alongside related Sb3GT1 crystal structures, and supported by theoretical calculations.Furthermore, they have successfully generated mutants with altered sugar selectivity via protein engineering, thereby expanding the boundaries of understanding of this novel enzyme family.The unique apiosyltransferase discovery and subsequent characterization are well executed, notably identifying the critical RLGSDH motif involved in substrate recognition.The authors have also pioneered a de novo biosynthesis in Nicotiana benthamiana to produce liquiritin apioside, a significant advancement in this field.Overall, this paper is well-constructed, clearly laying out the logic underpinning the enzyme characterization, supported by compelling results and robust data.
However, it is important to note that a preprint paper titled "Discovery of the apiosyltransferase, celery UGT94AX1 that catalyzes the biosynthesis of a flavone glycoside, apiin" was posted in bioRxiv very recently.This paper similarly claims the identification of a plant apiosyltransferase, in this case, celery UGT94AX1 (AgApiT), and there seems to be a significant overlap in the content of the two papers.Thus, it would be beneficial to revise sections such as lines 74-76, 360-363, and 435-436 to emphasize the unique features of GuApiGT in comparison to AgApiT.Highlighting these distinctions will reinforce the novelty of your study.
Nevertheless, the discovery of GuApiGT remains intriguing and constructive.There are, however, several points that I believe warrant attention and further clarification: Major Revisions: 1.It seems unusual that enzyme kinetic studies are not part of your enzyme characterization.For novel enzymes such as GuApiGT, key kinetic parameters, like KM, Vmax, and kcat, are essential to understand its behavior.Such studies could yield valuable insights into the enzyme's reaction efficiency and substrate affinity.I would strongly recommend adding an enzyme kinetics study to strengthen the thoroughness of your characterization.
2. It would be beneficial to provide context for the selection of the 15 reported UGTs in Supplementary Fig. 1.If there's a specific reason for this choice, or it's based on a review or database, providing this information would aid comprehension.Additionally, the significance of the purple font is unclear.
3. In Fig. 2a, it appears the LC peak may be truncated or edited.Please revisit your original data to verify the accuracy of this representation.
4. Unlike the engineered Sb3GT1, GuApiGT accepts flavonoid, lignan, or coumarin glycosides, but not free aglycones.An explanation based on structural evidence would be enlightening.Moreover, an explanation for the regioselectivity of GuApiGT towards 5-O-glycosides instead of 3-O-glycosides, as informed by the solved structures, would be informative.

5.
While obtaining crystal structures is commendable, a comparison of GuApiGT with other GTs to understand why they cannot perform apiosyltransferase functions would be very insightful.6. Performing enzymatic activity assays with the mutant enzyme in the presence of UDP-Xyl and UDP-Glc would offer direct evidence of substrate turnover, greatly reinforcing your claim of substrate binding.
7. The significant ΔΔE for GLU272 warrants its inclusion in mutagenesis studies.Also, the significance of the bold font in Supplementary Table 7 should be clarified.Lastly, consistency in amino acid notation between the main text and supplementary information is necessary.

Responses to Reviewers' comments
Reviewer #1: Wang et al discovered the first apiosyltransferase, GuApiGT, from the medical plant Glycyrrhiza uralensis and characterized its catalytic mechanism through crystallography, mutagenesis, phylogenetic analysis, and plentiful calculations.The conclusions are convincing and were further verified by applied mutagenesis in GuApiGT and heterologous glycosyltransferases. Based on the newly gained apiotransferases and knowledges, the authors built up de novo biosynthetic pathway of several bioactive apiosides in Nicotiana benthamiana.The findings are novel and practical in the biosynthesis of pharmaceutically important apiosides.

R:
We thank the reviewer for carefully reading our manuscript, and giving us the valuable suggestions and comments.
Here are some small questions: 1) Line 281-283, "The peptide L135-E156 also changed remarkably, probably due to the hydrogen bond interaction with glucose 6-OH after I136T mutation" It was not clearly explained here.Did the authors mean the increasement in molecular weight derived from the hydrogen-deuterium exchange between 136T and glucose 6-OH?This hypothesis looks unreasonable.

R:
The increasement in molecular weight is not derived from the exchange between T136 and glucose 6-OH.Instead, it is derived from the hydrogen-deuterium exchange between the protein (hydrogen atoms at the outer surface of the protein) and the solvent D2O.After mutagenesis, the increasement in molecular weight of certain peptides may be different for the wild type and the mutant, as mutagenesis may change conformation of the protein.The hydrogen-deuterium exchange mass spectrometry (HDX-MS) technique was firstly published in 1993 (J.Am.Chem.Soc.1993, 115, 6317-6321; Protein Sci.1993, 2, 522-531).Currently, it has been developed into a popular tool to dissect the catalytic mechanisms of enzymes (Chem.Rev. 2022, 122, 7562-7623) (Fig. R1).In the present work, the molecular weight of peptide L135-E156 increased by around 2 Da when I136 was mutated to threonine (Fig. 5e).This result demonstrated the significance of T136 in recognizing UDP-Glc.To avoid any ambiguity, we have revised this sentence into "The L135-E156 peptide of I136T/L369/H373Q mutant exhibited remarkable increase of deuterium uptake, verifying the significance of T136 in recognizing UDP-Glc (Supplementary Figures137).". 2) Line 304-307, the authors stated that the declined catalytic efficiency of mutants come from the increased flexibility of the NTD, which has low affinity to glycoside.
Actually, L369/H373Q, G370/H373Q and I136T/G370/H373Q mutants displayed comparable catalytic efficiency to WT, suggesting the mutagenesis impair their affinity to UDP-sugar rather than the substrate binding capacity.Please give more reasonable explain to the results.Besides, "substrate" in this paragraph should be "glycoside" or "sugar acceptor".e enzyme, UDP-Api as sugar donor).The native enzyme showed higher activities than the mutants under the same reaction conditions (Fig. R2).
Generally, the C-terminal domain (CTD) and N-terminal domain (NTD) of UGTs are responsible for sugar donor binding and sugar acceptor binding, respectively (Biotechnol.Adv.2022, 60, 108030).In the present work, HDX-MS analysis indicated increased flexibility of the NTD in the mutant enzymes, which may cause decreased affinity between the sugar acceptor and the enzyme.These deductions were consistent with enzyme assay results.
In addition, we have revised "substrate binding" to "sugar acceptor" in this manuscript.
3) Line 318-338, although the Sb3GT1 375S/Q377H mutant obtained larger UDP-sugar binding motif, it can only recognize UDP-Api in a very low efficiency.Is there some explain for that?The authors concluded that R368 of GuApiGT is key residue in the R: For wild type Sb3GT1, F373 has π-π interactions with kaempferol (Fig. R3).While the 375S/Q377H mutant could recognize UDP-Api, the introduction of S375 led to the loss of interactions between the binding pocket and the sugar acceptor.Thus, the apiosylation activity of the mutant was low.
These results have been included into Figure 6b.4) Line 523-524, "The mobile phase was a gradient elution of solvents A (acetonitrile, ACN) and B (water containing 0.1% formic acid)", the solvents are different from that in Supplementary Table 4.

R:
We are sorry for this mistake.The description in the Supplementary Table 4 is correct.
We have corrected this mistake.

Reviewer #2:
The manuscript entitled 'Discovery, mechanisms, and engineering of the missing apiosylation step in the biosynthesis of flavonoid aposides in Leguminosae plants' reported an apiosyltransferase GuApiGT from medicinal plant G. uralensis.
Biochemical characterization supported that GuApiGT specifically utilizes UDPapiose as a sugar donor and catalyzes 2-O-apiosylation of flavonoid glycosides.The binding site for UDP-apiose was determined through structural determination, theoretical calculations, and metagenesis analysis.This enzyme also shows broad substrate promiscuity, it accepts 37 glycosides of flavonoids, liganans, and coumarins.
Several new compounds were generated and may show bioactivity.The authors successfully performed de novo biosynthesis to produce various flavonoid apiosides in tobacco.This work discovered 121 candidate apiosyltransferase genes from Leguminosae plants and determined the first crystal structure of apiosyltransferase.The key apiose binding motif (RLGSDH) was identified, providing important information for engineering of glycosyltransferases.The results possess significant novelty, the presentation is readable.Below are some of my major and minor concerns regarding the article: R: We thank the reviewer for carefully reading our manuscript, and giving us the valuable suggestions and comments.
Major Concern: 1.The authors mention many times that 'no reported UGTs could accept UDP-apiose as the sugar donor'; however, there is a research article, Identification of an apiosyltransferase in the plant pathogen Xanthomonas pisi (PLoS ONE 13(10) 2018), reported the first evidence of an apiosyltransferase (XpApiT).Although the sugar acceptor of XpApiT was unknown, indirect activity assay (UDP-Glo) and in microbe co-expression supported that XpApiT specifically utilizes UDP-apiose as sugar donor.
Does XpApiT possess the conserved apiose-binding residues or motifs, such as RLGSDH motif?Please rephrase the relevant sentences, 'no reported……', into more appropriate ones.
R: Thank you very much for this constructive comment.We did have noticed XpApiT (PLoS One, 2018, 13, e0206187).While it may be a potential apiosyltransferase, the authors did not characterize structure of the enzyme catalysis product.
Following the reviewer's suggestion, we analyzed the sequence information of XpApiT, but did not find the RLGSDH motif characteristic for Leguminosae ApiGTs discovered in our study.In fact, we did not find the conserved PSPG box for UGTs (Fig. R5).We want to mention that the Science paper was published after the initial submission of our paper (Mar 13, 2023, NATCATAL-23030347).Therefore, we did not read this paper when we were preparing our manuscript.
Based on the above situations, we have revised our statements in the manuscript into "To our best knowledge, GuApiGT is the first phenolic apiosyltransferase that has been reported."As no evidences prove that UGT73CY2 could accept phenolic compounds as substrate, it should be reasonable to state that GuApiGT is the first apiosyltransferase that can utilize phenolic compounds.We have also compared the sequences of GuApiGT and the other ApiGTs.H18 and D115 are highly conserved (Fig. R7).We have added descriptions on their roles in the manuscript: "During the process to form the glycosidic bond between O2′′ of 2 and C1′′′ of UDP-Api, H18 could partially deprotonate 2, with the assistance of D115.Once the reaction is completed, D115 is protonated in the product complex.".R: We had used the PDB atom labelling (C1 and O5) in the previous version.To avoid any confusion, we used C1 and O2 in the revised manuscript (Fig. 4f).
6.The 'Number of atoms' of Sb3GT1 375S/Q377H in Table 8 does not match to the crystal structure.Please revise Table 8.

R:
We are sorry for this mistake.These data have been corrected in Table 8.However, it is important to note that a preprint paper titled "Discovery of the apiosyltransferase, celery UGT94AX1 that catalyzes the biosynthesis of a flavone glycoside, apiin" was posted in bioRxiv very recently.This paper similarly claims the identification of a plant apiosyltransferase, in this case, celery UGT94AX1 (AgApiT), and there seems to be a significant overlap in the content of the two papers.Thus, it would be beneficial to revise sections such as lines 74-76, 360-363, and 435-436 to emphasize the unique features of GuApiGT in comparison to AgApiT.Highlighting these distinctions will reinforce the novelty of your study.
Nevertheless, the discovery of GuApiGT remains intriguing and constructive.There are, however, several points that I believe warrant attention and further clarification: R: We thank the reviewer for carefully reading our manuscript, and giving us the valuable suggestions and comments.We have also noticed the preprint paper about Second, the sequence of UGT94AX1 has a 44-amino acid PSPG box, and it does not contain the RLGSDH motif (Fig. R8b).Therefore, these features may be unique for Leguminosae apiosyltransferases.GuApiGT and the other four ApiGTs represent a novel group of glycosyltransferases. Major Revisions: 1.It seems unusual that enzyme kinetic studies are not part of your enzyme characterization.For novel enzymes such as GuApiGT, key kinetic parameters, like Km, Vmax, and kcat, are essential to understand its behavior.Such studies could yield valuable insights into the enzyme's reaction efficiency and substrate affinity.I would strongly recommend adding an enzyme kinetics study to strengthen the thoroughness of your characterization.
R: UDP-apiose is unstable and is commercially unavailable.This is the reason why we did not present the enzyme kinetic parameters of GuApiGT.In our study, we introduced the UDP-apiose/UDP-xylose synthase (UAXS) from Arabidopsis thaliana into the enzyme catalysis system, which could provide UDP-apiose as sugar donor.
During this revision, we managed to obtain purified UDP-apiose.UAXS could convert UDP-glucuronic acid (UDP-GlcA) into both UDP-apiose and UDP-xylose.We used the method published by Tae Fujimori et al. (Carbohydr.Res.2019, 477, 20-25.)and UAXS.A total of 30 parallel tubes were used.The reactions were performed at 25°C for 4h and then centrifuged at 15,000 rpm for 30 min.The products were subsequently purified by reversed-phase HPLC.HPLC was performed on an Inertsustain AQ-C18 ( 5μm, 4.6 × 250 mm column; GL Sciences, Tokyo, Japan) at a flow rate of 1.0 mL/min.
The conversion rates in percentage were calculated from HPLC peak areas of glycosylated products and substrates.Michaelis-Menten plot was fitted.
2. It would be beneficial to provide context for the selection of the 15 reported UGTs in Supplementary Fig. 1.If there's a specific reason for this choice, or it's based on a review or database, providing this information would aid comprehension.Additionally, the significance of the purple font is unclear.

R:
We selected 15 reported UGTs with different sugar donor selectivity for the phylogenetic analysis (Fig. R10).However, they were mainly clustered based on substrate type and glycosylation site rather than sugar donor selectivity.GuApiGT was clustered with two glycosides 2-OGT (ZjOGT38 and TcOGT4).For better understanding, we have prepared a new phylogenetic analysis figure in the revised manuscript (Supplementary Fig. 1).Supplementary Fig. 1 Phylogenetic analysis of GuApiGT (MSTRG.23171.4)with reported UGTs using MEGA6 software with the maximum likelihood method.The bootstrap consensus tree inferred from 1,000 replicates was taken to represent the evolutionary history of the taxa analyzed.All the GenBank accession numbers used in this study are listed in Supplementary Table 2. 3.In Fig. 2a, it appears the LC peak may be truncated or edited.Please revisit your original data to verify the accuracy of this representation.

R:
The original HPLC chromatograms were provided in Fig. R11.The peaks at around 2 min were proteins, and these peaks were not related with the catalytic function.For better understanding, we only provided the chromatograms between 4-12 min in Fig. 2a.
Fig. R11 a,b, HPLC analysis of GuApiGT catalyzed product using 1 as the substrate.c,d, HPLC analysis of GuApiGT catalyzed product using 2 as the substrate.UDP-Api was produced by adding UDP-GlcA, purified UAXS, and NAD + to the mixed system.
The chromatographic peak with a retention time of around 2 min is the protein peak.
4. Unlike the engineered Sb3GT1, GuApiGT accepts flavonoid, lignan, or coumarin glycosides, but not free aglycones.An explanation based on structural evidence would be enlightening.Moreover, an explanation for the regioselectivity of GuApiGT towards 5-O-glycosides instead of 3-O-glycosides, as informed by the solved structures, would be informative.
The active site of GuApiGT shapes like a "hammer".Compound 2 can fit this shape very well, but its aglycone isoliquiritigenin (2) only fits the handle region of this "hammer".Thus, 2 is not stable in the active pocket.We further conducted 100-ns MD simulations of GuApiGT/UDP-Api/2 and GuApiGT/UDP-Api/2.The MM/GBSA binding free energy of 2 (-58.7 ± 5.3 kcal/mol) in the active pocket is significant higher than 2 (-78.8 ± 4.2 kcal/mol).These data explained why GuApiGT could not accept GuApiGT does not have this function.This new enzyme will be published elsewhere in the future.We believe more mechanisms will be interpreted as more ApiGTs are discovered.

R:
We have analyzed all the crystal structures of reported GTs.We found that a part of UDP-sugar binding region of GuApiGT is remarkably different from other GTs.This region is composed of the R 368 L 369 G 370 S 371 loop and the start of α helix (D 372 H 373 ), which form a large secondary structure compared with other GTs (Fig. 4d).Our study also proved the significance of this motif for sugar donor selectivity by structural analysis and site-directed mutagenesis.5.In Figures 4e and 4g, the rationale behind presenting some residues as lines and some as sticks is not evident.Furthermore, the coloring of R368 in orange is not explained in the figure legend.

R:
We have revised Figures 4e and 4g, and have added detailed information in the legend.dashes, and magenta dashes, respectively.The MM region atoms are depicted using lines.
6.The term "directed evolution" might not accurately describe your engineering efforts.
Perhaps "rational design" or "protein engineering" would be more fitting.

R:
We have revised it into "protein engineering".

Fig. R3
Fig. R3 The key binding motif in Sb3GT1/UDP and Sb3GT1 375S/Q377H mutant/UDP-Glc.The key amino acids were labeled by red color font.Kaempferol was docked into Sb3GT1 and its mutant based on the crystal structure of VvGT1 (PDB ID: 2C1Z).The hydrogen-bond interaction and π-π interaction are shown as yellow and purple dashes, respectively.

Fig. 4e A
Fig. 4e A representative configuration of GuApiGT/UDP-Api/2 extracted from MD simulations.The hydrogen-bond interactions and π-π/cation-π interactions are shown as yellow and purple dashes, respectively.The key amino acids interacted with ligands are highlighted using sticks.The unique R368 is depicted as orange sticks, the others as blue.The other amino acids in key motif are depicted using lines.

Fig. 4g 5 .
Fig. 4g QM/MM optimized geometry of transition state (TS) at the theory of B3LYP/6-311++g(2d,2p): amber with the electronic embedding scheme and thermal zero-point energy calculated from the theory of B3LYP/6-31g(d): amber.The QM region atoms, hydrogen bonds, and key angle and distances are highlighted in green sticks, yellow dashes, and magenta dashes, respectively.The MM region atoms are depicted using UGT94AX1 from celery (https://doi.org/10.1101/2023.05.22.541790,Posted May 23, 2023).Our manuscript was posted on the preprint website Research Square on March 29, 2023.Because our manuscript was open to the public two months earlier than the UGT94AX1 paper, we believe that GuApiGT is the first phenolic apiosyltransferase that has been characterized.It is interesting to discover UGT94AX1 as an apiosyltransferase from celery (Apium graveolens) of the Apiaceae family.We have compared the catalytic features of UGT94AX1 and GuApiGT.They show remarkably different features.First, UGT94AX1 shows low catalytic activity, with Km (affinity) and kcat/Km (catalytic efficiency) values of 15 μmol•L -1 and 5.8*10 -5 s -1 •μmol -1 •L, respectively.(Fig. R8).In contrast, these values for GuApiGT were 2.59 μmol•L -1 and 4.2*10 -2 s -1 •μmol -1 •L.The catalytic efficiency (kcat/Km) was around 1000-fold different.
free aglycones as sugar acceptor.Compound 20 (5-O-glycoside) shares a similar binding mode with compound 2, while compound 38 (3-O-glycoside) exhibits great steric hindrance in fitting the handle region.The above descriptions have been added in Supplementary Figure 141.Supplementary Fig. 141 Binding modes of compounds 2 (thick green sticks), 2 (thick yellow sticks), 20 (thick magenta sticks) and 38 (thick cyan sticks) in GuApiGT.UDP-Api and important residues are depicted as thick pink sticks and grey thin sticks, respectively.The shape of the binding pocket is depicted by surface with the transparency of 0.4.a, top view; b, front view.Given the very limited number of apiosyltransferases that have been reported, it is hard to intensively interpret their catalytic mechanisms.Recently, we have discovered a new ApiGT, which could catalyze the apiosylation of flavone 3-O-glycosides (Fig.R12).

Fig. R12
Fig. R12 Functional characterization and substrate promiscuity of a new ApiGT which can catalyze flavone 3-O-glycosides.

Fig. 4e A
Fig. 4e A representative configuration of GuApiGT/UDP-Api/2 extracted from MD simulations.The hydrogen-bond interactions and π-π/cation-π interactions are shown as yellow and purple dashes, respectively.The key amino acids interacted with ligands are highlighted using sticks.The unique R368 is depicted as orange sticks, the others as blue.The other amino acids in key motif are depicted using lines.

Fig. 4g
Fig. 4g QM/MM optimized geometry of transition state (TS) at the theory of B3LYP/6-311++g(2d,2p): amber with the electronic embedding scheme and thermal zero-point energy calculated from the theory of B3LYP/6-31g(d): amber.The QM region atoms, hydrogen bonds, and key angle and distances are highlighted in green sticks, yellow

7 . 8 .
A clearer statement of the structural superimposition could be "For easy understanding, we have revised this sentence into "The RLGSDH (368-373) motif in GuApiGT is mapped to FFGDQ (372-376) of Sb3GT1.".The error bar in Figure5cappears inconsistent with others.Please review and rectify if necessary.R:We have revised error bars in all the figures according to the journal instructions.

9 .
The notation "375S" could potentially confuse the readers.I recommend providing further clarification in line 327, 330, Figure6c, and Supplementary Figure148.R: To avoid any confusion, we have added this description "Based on structural analysis, we inserted a serine residue into the motif and constructed the 375S/Q377H mutant of Sb3GT1, as well as the F372R/Q376H and F372R/375S/Q377H mutants.". 10.The differences in visualization methods used in Fig.6a and 6c(sticks vs. lines) and the presence of a white cartoon in the zoomed image of Fig. 6a should be adequately justified in the figure legends.R: Both the sticks in Fig.6a and the lines in Fig.6c represent amino acids.The reason for using lines is due to clearly show the key motif and the comparison between native and 375S/Q377H.We have improved the figure legends for Figures 6a and 6c according to the reviewer's suggestion.

Fig. 6 a
Fig. 6 a, The crystal structure of Sb3GT1 (PDB ID: 8IOE) and the FFGDQ (372-376) motif.The image on the right is an enlargement of the dashed rectangle, where the red part represents CTD and the grey part represents NTD.The amino acids in key motif of Sb3GT1 are highlighted using sticks.c, The crystal structure of Sb3GT1375S/Q377H mutant (PDB ID: 8IOD) and superimposition of its key motif to that in wild type.The amino acids in key motif of 375S/Q377H mutant are depicted using lines.
Very recently, Anne Osbourn's group from John Innes Centre reported the first characterized apiosyltransferase UGT73CY2(Science, 2023, 379, 1252-1264.Mar 23,   2023).This enzyme could catalyze the apiosylation of one triterpenoid saponin.Besides this, very limited information on the catalytic feature of UGT73CY2 was provided in this paper.It is not known whether UGT73CY2 could catalyze other types of substrates, or whether it specifically accepts UDP-Api.Its sequence contains the PSPG box, but not the RLGSDH motif.Thus, GuApiGT and the other four Leguminosae ApiGTs represent a novel group of apiosytransferase.

Table 8 .
Data collection and refinement statistics of Sb3GT1 crystals.
a Values in parentheses are for highest-resolution shell.
In this work, we report the discovery of GuApiGT, the first apiosyltransferase derived from the medicinal plant Glycyrrhiza uralensis."R: We have revised this sentence into "We identified the first phenolic apiosyltransferase GuApiGT from Glycyrrhiza uralensis.".We have added reference 2 "Ann Chim Phys 1843, 9, 250-252." to line 42.Reference 1 has been added to lines 50-52.Line 71 has cited References 14. References 43 and 44 have been added to line 389 (line 370, previous version).The fresh plant was collected by the authors from Inner Mongolia Autonomous Region of China, which is the traditional cultivation region for G. uralensis.The plant species was identified as Glycyrrhiza uralensis Fisch.but not closely related species, according to the ITS2 sequence by a DNA barcoding method established by our group (Song W, et al.Anal.Chem.2017, 89, 3146-3153) (Fig. R13).
R:3.In lines 57-62, the identification of Glycyrrhiza uralensis Fisch.as the target plant could be more solidly substantiated, considering the existence of multiple popular medicinal plants within the Leguminosae family.Also, the discussion about liquiritin apioside could be made more relatable to lines 84-86.R:

Table R1 .
Raw data for the determination of kinetic parameters